Anti-BubR1 antibody [8G1]

• Supplier: Abcam
• Catalog: ab4637
• Prices: $400.00
• Sizes: 200 µg
• Host: Mouse
• Clonality: Monoclonal
• Isotype: IgG1
• Clone: 8G1
• Applications: IP ICC/IF ICC/IF WB
• Species Reactivities: Human
• Antigen: N-terminal tagged fusion recombinant fragment, corresponding to amino acids 1-350 of Human BubR1
• Description: Mouse Monoclonal
• Gene: BUB1B
• Conjugate: Unconjugated

Product images

Immunofluorescent imaging of an asynchronous cycling population of human cells (U2OS) with ab4637 strikingly confirms the specificity of this antibody.  No signal is detected from interphase cells, whereas cells undergoing mitosis accumulate BubR1 at the kinetochores.  Image reveals kinetochores at prometaphase, and exactly agrees with numerous published images of BubR1 (see for example Johnson et al J Cell Sci. 117(Pt 8):1577-89 (2004). IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primaryantibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes.  All blocking and incubation steps carried out at 37 degrees C.

HeLa cells were stained with anti-BubR1 (ab4637) in green and DAPI in blue in panel one and anti-BubR1 (green) and SH-CREST (red) to stain the centromeres in panel 2. Fix cells for 30 minutes on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 min. with 1 mg/ml Na borohydride OR 100 mM ammonium chloride in PEM. Permeablize 30 min. with 0.5% TX-100 in PEM. Block 30 minutes in 5% milk in TBST. Primary antibody was incubated 1/200 overnight at 4oC diluted in 5% milk in TBST. Secondary antibody was incubated for 1 hour at RT diluted in 5% milk in TBST. Post-fix 20 min. on ice in 4% formaldehyde in PEM. Quench autofluorescence 2 x 5 min. with ammonium chloride in PEM. Counterstain with DAPI in TBST. Mount with ProLong Gold antifade reagent from Invitrogen. Notes: Ample washing between each step. TBST = Tris buffered saline + 0.1% Tween. PEM = 80 mM K-PIPES, pH 6.8, 5 mM EGTA, 2 mM MgCl2.

ab4637 staining HeLa cells by ICC/IF.  Cells were PFA fixed and permeabilzed in 0.5% CHAPS prior to blocking with 20% serum for 1 hour at 23°C.  The primary antibody was diluted 1/100 in 5% Goat Serum/Mops Buffered Saline and incubated with the sample for 1 hour at 23°C.  A FITC conjugated goat anti-mouse was used as the secondary.  In this application, ab4637 demonstrates that BUBR1 is localized to the kinetochores of mitotic chromosomes in prophase and prometaphase, but not in other mitotic phases.See Abreview

Immunofluorescence of nocodazole treated HeLa cells with ab4637 staining BubR1 (green). CREST was used to mark centromeres (red) and the DNA is stained with DAPI (blue).Cells were fixed with PFA 4% and permealized with Triton X-100 0.1%. Blocking was done with 4% BSA. ab4637 was used at a 1:200 dilution in 4% BSA for 1.5h at room temperature. Secondary antibodies were incubated for 45min. Coverslips were then stained with DAPI and mounted with Mowiol mounting media.ab4637 reveals BubR1 in mitotic cells colocalizing with kinetochores, as expected.

Product References

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